This proposal aims to determine the contribution of the complement system to pathology in an alpha- synuclein based, mouse model of Parkinson disease (PD). Our lab has pursued the idea that alpha-synuclein, an intracellular protein abnormally aggregated in PD brains, is a trigger for innate immune system activation associated with PD. We have shown that targeted overexpression of alpha-synuclein in the substantia nigra of mice driven by an adeno-associated virus vector (AAV-SYN) recapitulates the microgliosis and slow progressive cell death observed in human PD. Additionally, knocking out microglial Fc-gamma-receptors reduces alpha-synuclein induced neuronal degeneration, suggesting an innate immunity-based disease mechanism. The proposed research attempts to build upon this characterization, by investigating the hypothesis that activation of the complement cascade is required for mediating the dopaminergic neurotoxicity of alpha-synuclein in vivo. First, we will determine whether alpha synuclein can lead to complement activation in our AAV-SYN mouse model of PD. We will measure expression of complement system proteins and mRNAs in mice injected with AAV-SYN or a control AAV vector (AAV-GFP) at 2 and 4 weeks post-injection. Next, we will determine the effect of alpha-synuclein on microglial expression of complement components and receptors, and microglial effector function. We will measure changes in mRNA and protein expression of complement components expressed by microglia, changes in surface complement receptors by flow cytometry, changes in phagocytosis by commercially available assays, and changes in cytokine expression by ELISA. Lastly, we will test the rationale that blocking the complement system will reduce inflammation and neurodegeneration associated with alpha-synuclein overexpression. We will inject both wild type mice and mice expressing a complement inhibitor (Crry) under an astrocyte-specific promoter with AAV-GFP and AAV- SYN in the substantia nigra. We will assess dopaminergic neuron loss at 6 months post-injection with unbiased stereology, microglial activation at 4 weeks post-injection through immunofluorescence staining and confocal microscopy, and cytokine expression at 2 and 4 weeks post-injection through previously characterized qPCR. The proposed training plan is sponsored by Dr. David Standaert. The overall goal of the training plan is to provide the PI with a solid foundation for a successful career as a physician scientist. Included in the training plan are experiences that help the PI: 1) gain competence in a variety of techniques integrating neurobiology and immunology, 2) collaborate with other scientists, 3) develop hypothesis-driven research, 4) present data in a written and oral format, 5) effectively integrate research with clinic, and 6) responsibly conduct research. Relevance to Public Health: Parkinson disease is the second most common neurodegenerative disorder, and the risk of disease increases with age. At least 3% of the United States population above age 65 is diagnosed with Parkinson disease. However, there are currently no neuroprotective treatments for PD. This proposal aims to provide evidence for a role for the complement system in PD pathology. This study would serve as rationale for the complement system as an innovative target for the development of PD therapies and biomarkers.